Anti-human papillomavirus 16 e6 t cell receptors

ABSTRACT

Disclosed is a T cell receptor (TCR) having antigenic specificity for an HLA-A2-restricted epitope of human papillomavirus (HPV) 16 E6, E629-38. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells are also provided. Antibodies, or an antigen binding portion thereof, and pharmaceutical compositions relating to the TCRs of the invention are also provided. Also disclosed are methods of detecting the presence of a condition in a mammal and methods of treating or preventing a condition in a mammal, wherein the condition is cancer, HPV 16 infection, or HPV-positive premalignancy.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/846,167, filed Jul. 15, 2013, which is incorporatedherein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 45,955 Byte ASCII (Text) file named“716827ST25.TXT,” dated May 27, 2014.

BACKGROUND OF THE INVENTION

The primary cause of some cancer types such as, for example, uterinecervical cancer, is human papillomavirus (HPV) infection. Despiteadvances in treatments such as chemotherapy, the prognosis for manycancers, including HPV-associated cancers, may be poor. Accordingly,there exists an unmet need for additional treatments for cancer,particularly HPV-associated cancers.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a T cell receptor (TCR) havingantigenic specificity for human papillomavirus (HPV) 16 E6 andcomprising a human variable region and a murine constant region.

Another embodiment of the invention provides an isolated or purified TCRhaving antigenic specificity for HPV 16 E6 and comprising the amino acidsequences of SEQ ID NOs: 3-8.

The invention further provides related polypeptides and proteins, aswell as related nucleic acids, recombinant expression vectors, hostcells, and populations of cells. Further provided by the invention areantibodies, or antigen binding portions thereof, and pharmaceuticalcompositions relating to the TCRs (including functional portions andfunctional variants thereof) of the invention.

Methods of detecting the presence of a condition in a mammal and methodsof treating or preventing a condition in a mammal, wherein the conditionis cancer, HPV 16 infection, or HPV-positive premalignancy, are furtherprovided by the invention. The inventive method of detecting thepresence of a condition in a mammal comprises (i) contacting a samplecomprising cells of the condition with any of the inventive TCRs(including functional portions and functional variants thereof),polypeptides, proteins, nucleic acids, recombinant expression vectors,host cells, populations of host cells, antibodies, or antigen bindingportions thereof, or pharmaceutical compositions described herein,thereby forming a complex, and (ii) detecting the complex, whereindetection of the complex is indicative of the presence of the conditionin the mammal, wherein the condition is cancer, HPV 16 infection, orHPV-positive premalignancy.

The inventive method of treating or preventing a condition in a mammalcomprises administering to the mammal any of the TCRs (includingfunctional portions and functional variants thereof), polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs(including functional portions and functional variants thereof),polypeptides, proteins described herein, or any host cell or populationof host cells comprising a recombinant vector which encodes any of theTCRs (including functional portions and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a bar graph showing expression of HPV 16 E6 (white bars), HPV16 E7 (shaded unhatched bars), HPV 18 E6 (unshaded hatched bars), or HPV18 E7 (shaded hatched bars) relative to glyceraldehyde 3-phosphatedehydrogenase (GAPDH) expression by CaSki cells, HeLa cells, 624 cells,or cells from tumor 3809.

FIGS. 2A and 2B are bar graphs showing interferon-gamma (IFN-γ) (pg/mL)secreted by tumor infiltrating lymphocytes (TIL) from fragments (F) 2-14of tumor 3809 (A), fragments 15-24 (B) of tumor 3809, or melanoma TILupon co-culture with autologous dendritic cells (DCs) which had beenpulsed with HPV 16 E6 alone (white bars), HPV 16 E6 in combination withanti-class I antibody (shaded unhatched bars), HPV 16 E7 alone (unshadedhatched bars), HPV 16 E7 in combination with anti-class I antibody(shaded hatched bars), gp100 (black bars), or OKT3 (horizontal stripedbars).

FIG. 3 is a bar graph showing IFN-γ (pg/mL) secreted by expanded numbersof 3809 4-1BB-selected TIL upon co-culture with 293-A2 cells (HEK-293cells with stable expression of HLA-A2) transfected with greenfluorescent protein (GFP), 293-A2 cells transfected with HPV 16 E6, 3809lymphoblastoid cell line (LCL) (B cells that have been transformed usingEpstein-Barr virus) cultured without peptide, 3809 LCL co-cultured withan HPV 16 E6 peptide pool, or OKT3.

FIG. 4A is a bar graph showing IFN-γ (pg/mL) secreted by peripheralblood lymphocytes (PBL) that were not transduced (untransduced)(unshaded bars) or transduced with a nucleotide sequence encoding SEQ IDNOs: 17 and 18 (E6 TCR; shaded bars) upon co-culture with target 293-A2cells pulsed with HPV 16 E6₂₉₋₃₈ peptide, 293-A2 cells pulsed with HPV16 E7₁₁₋₁₉ peptide, 293-A2 cells transduced with a plasmid encoding HPV16 E6, 293-A2 cells transduced with a plasmid encoding GFP, 293 cellstransduced with a plasmid encoding HPV 16 E6, 624 cells transduced witha plasmid encoding HPV 16 E6, 624 cells transduced with a plasmidencoding HPV 16 E7, SCC152 cells, SCC90 cells, CaSki cells, HPV-18cervical cancer cells, melanoma control cells, cholangio control cells,624 cells, or SiHa cells. HLA-A2 and HPV-16 expression by each targetcell is indicated in the bottom of FIG. 4A (“+” indicates positive forexpression and “−” indicates negative for expression).

FIG. 4B is a bar graph showing IFN-γ (pg/mL) secreted by PBL transducedwith a nucleotide sequence encoding SEQ ID NOs: 17 and 18 uponco-culture with target 293-A2 cells transduced with a plasmid encodingHPV 16 E6, 293 cells transduced with a plasmid encoding HPV 16 E6, SCC90cells, CaSki cells, 624 cells, DMF/624 cells, or 4.7.20 cells pulsedwith HPV 16 E7 peptides with no antibody (black bars), anti-MHC Class Iantibody (grey bars), or anti-MHC Class II antibody (unshaded bars).

FIG. 5 is a bar graph showing IFN-γ (pg/mL) secreted by untransducedCD8-positive PBL (unshaded unhatched bars), untransduced CD4-positivePBL (cross-hatched unshaded bars), or PBL (CD8 positive (E6 TCR; shadedunhatched bars) or CD4 positive (E6 TCR; cross-hatched shaded bars))that were transduced with a nucleotide sequence encoding SEQ ID NOs: 17and 18 upon co-culture with target 293-A2 cells pulsed with HPV 16E6₂₉₋₃₈ peptide, 293-A2 cells pulsed with HPV 16 E7₁₁₋₁₉ peptide, 293-A2cells transduced with a plasmid encoding HPV 16 E6, 293-A2 cellstransduced with a plasmid encoding GFP, 293-A2 cells, 624 cellstransduced with a plasmid encoding HPV 16 E7, 624 cells transduced witha plasmid encoding HPV 16 E6, SCC152 cells, SCC90 cells, CaSki cells,HPV-cervical cancer cells, melanoma control cells, 624 cells, or SiHacells.

FIG. 6A is a bar graph showing IFN-γ (pg/mL) secreted by untransduced(UT) CD8-positive PBL (unshaded bars) or CD8 positive PBL transducedwith a nucleotide sequence encoding SEQ ID NOs: 17 and 18 (shaded bars)upon co-culture with target T2 cells pulsed with varying concentrationsof E6₂₉₋₃₈ peptide (−log M).

FIG. 6B is a bar graph showing IFN-γ (pg/mL) secreted by untransducedCD4-positive PBL (unshaded bars) or CD4 positive PBL transduced with anucleotide sequence encoding SEQ ID NOs: 17 and 18 (E6 TCR; shaded bars)upon co-culture with target T2 cells pulsed with varying concentrationsof E6₂₉₋₃₈ peptide (−log M).

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a T cell receptor (TCR), andfunctional portions and functional variants thereof, having antigenicspecificity for human papillomavirus (HPV) 16 E6 and comprising a humanvariable region and a murine constant region. In an embodiment of theinvention, the TCR has antigenic specificity for HPV 16 E6₂₉₋₃₈.

The HPV 16 is the subtype of HPV that is most commonly associated withmalignancy. Without being bound to a particular theory or mechanism, HPV16 is believed to cause cancer at least partly through the actions ofthe oncoprotein E6, which deregulates cell cycle control. HPV 16 E6 isconstitutively expressed in cancer cells and is not expressed by normal,uninfected human tissues. HPV 16 E6 is expressed in a variety of humancancers including, but not limited to, cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, and penis.

The TCR may have antigenic specificity for any HPV 16 E6 protein,polypeptide or peptide. In an embodiment of the invention, the TCR hasantigenic specificity for a HPV 16 E6 protein comprising, consisting of,or consisting essentially of, the amino acid sequence of SEQ ID NO: 1.In a preferred embodiment of the invention, the TCR has antigenicspecificity for a HPV 16 E6₂₉₋₃₈ peptide comprising, consisting of, orconsisting essentially of, the amino acid sequence of TIHDIILECV (SEQ IDNO: 2).

In an embodiment of the invention, the inventive TCRs are able torecognize HPV 16 E6 in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR elicits an immune response upon binding to HPV 16 E6within the context of an MHC class I molecule. The MHC class I moleculecan be any MHC class I molecule known in the art, e.g., HLA-A molecules.In a preferred embodiment of the invention, the MHC class I molecule isan HLA-A2 molecule.

The TCRs (including functional portions and functional variants thereof)of the invention provide many advantages, including when expressed bycells used for adoptive cell transfer. Without being bound by aparticular theory or mechanism, it is believed that because HPV 16 E6 isexpressed by HPV 16-infected cells of multiple cancer types, theinventive TCRs (including functional portions and functional variantsthereof) advantageously provide the ability to destroy cells of multipletypes of HPV 16-associated cancer and, accordingly, treat or preventmultiple types of HPV 16-associated cancer. Additionally, without beingbound to a particular theory or mechanism, it is believed that becausethe HPV 16 E6 protein is expressed only in cancer cells, HPV 16-infectedcells, or HPV-positive premalignancy cells, the inventive TCRs(including functional portions and functional variants thereof)advantageously target the destruction of cancer cells, HPV 16-infectedcells, or HPV-positive premalignancy cells, while minimizing oreliminating the destruction of normal, non-cancerous, non-HPV-infected,and non-HPV-positive premalignant cells, thereby reducing, for example,by minimizing or eliminating, toxicity. Moreover, the inventive TCRsmay, advantageously, successfully treat or prevent HPV-positive cancersthat do not respond to other types of treatment such as, for example,chemotherapy alone, surgery, or radiation. Additionally, the inventiveTCRs provide highly avid recognition of HPV 16 E6, which may,advantageously, provide the ability to recognize unmanipulated tumorcells (e.g., tumor cells that have not been treated withinterferon-gamma, transfected with a vector encoding one or both of HPV16 E6 and HLA-A2, pulsed with the E6₂₉₋₃₈ peptide, or a combinationthereof).

The phrase “antigenic specificity,” as used herein, means that the TCRcan specifically bind to and immunologically recognize HPV 16 E6 withhigh avidity. For example, a TCR may be considered to have “antigenicspecificity” for HPV 16 E6 if T cells expressing the TCR secrete atleast about 200 pg/mL or more (e.g., 200 pg/mL or more, 300 pg/mL ormore, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mLor more, 1000 pg/mL or more, 5,000 pg/mL or more, 7,000 pg/mL or more,10,000 pg/mL or more, or 20,000 pg/mL or more) of interferon gamma(IFN-γ) upon co-culture with antigen-negative HLA-A2+ target cellspulsed with a low concentration of HPV 16 E6 peptide (e.g., about 0.05ng/mL to about 5 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, or 5ng/mL). Alternatively or additionally, a TCR may be considered to have“antigenic specificity” for HPV 16 E6 if T cells expressing the TCRsecrete at least twice as much IFN-γ as the untransduced peripheralblood lymphocyte (PBL) background level of IFN-γ upon co-culture withantigen-negative HLA-A2+ target cells pulsed with a low concentration ofHPV 16 E6 peptide. Cells expressing the inventive TCRs (includingfunctional portions and functional variants thereof) may also secreteIFN-γ upon co-culture with antigen-negative HLA-A2+ target cells pulsedwith higher concentrations of HPV 16 E6 peptide.

The invention provides a TCR comprising two polypeptides (i.e.,polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β)chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR,or a combination thereof. The polypeptides of the inventive TCR cancomprise any amino acid sequence, provided that the TCR has antigenicspecificity for HPV 16 E6.

In an embodiment of the invention, the TCR comprises two polypeptidechains, each of which comprises a human variable region comprising acomplementarity determining region (CDR)1, a CDR2, and a CDR3 of a TCR.In an embodiment of the invention, the TCR comprises a first polypeptidechain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:3 (CDR1 of α chain), a CDR2 comprising the amino acid sequence of SEQ IDNO: 4 (CDR2 of α chain), and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 5 (CDR3 of α chain), and a second polypeptide chaincomprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6(CDR1 of β chain), a CDR2 comprising the amino acid sequence of SEQ IDNO: 7 (CDR2 of β chain), and a CDR3 comprising the amino acid sequenceof SEQ ID NO: 8 (CDR3 of β chain). In this regard, the inventive TCR cancomprise any one or more of the amino acid sequences selected from thegroup consisting of SEQ ID NOs: 3-8. Preferably, the TCR comprises theamino acid sequences of SEQ ID NOs: 3-5 or SEQ ID NOs: 6-8. In anespecially preferred embodiment, the TCR comprises the amino acidsequences of SEQ ID NOs: 3-8.

In an embodiment of the invention, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 9 (the variable region of an α chain), SEQ ID NO: 10 (thevariable region of a β chain), or both SEQ ID NOs: 9 and 10. Preferably,the inventive TCR comprises the amino acid sequences of both SEQ ID NOs:9 and 10.

The inventive TCRs may further comprise a constant region derived fromany suitable species such as, e.g., human or mouse. In an embodiment ofthe invention, the inventive TCRs further comprise a murine constantregion. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 15 (the constant region of a murine α chain), SEQ ID NO: 16(the constant region of a murine β chain), or both SEQ ID NOs: 15 and16. In a preferred embodiment, the inventive TCRs are chimeric TCRscomprising both a human variable region and a murine constant region.

In an embodiment of the invention, the inventive chimeric TCR maycomprise a combination of a variable region and a constant region. Inthis regard, the TCR can comprise an alpha chain comprising the aminoacid sequences of both of SEQ ID NO: 9 (the variable region of a human αchain) and SEQ ID NO: 15 (the constant region of a murine α chain), abeta chain comprising the amino acid sequence of both of SEQ ID NO: 10(the variable region of a human β chain) and SEQ ID NO: 16 (the constantregion of a murine β chain), or all of the amino acid sequences of SEQID NOs: 9-10 and 15-16.

As used herein, the term “murine” or “human,” when referring to a TCR orany component of a TCR described herein (e.g., complementaritydetermining region (CDR), variable region, constant region, alpha chain,and/or beta chain), means a TCR (or component thereof) which is derivedfrom a mouse or a human, respectively, i.e., a TCR (or componentthereof) that originated from or was, at one time, expressed by a mouseT cell or a human T cell, respectively.

In an embodiment of the invention, the chimeric TCR can comprise an αchain of a TCR and a β chain of a TCR. Each of the α chain and β chainof the inventive chimeric TCR can independently comprise any amino acidsequence. Preferably, the α chain comprises the human variable region ofan α chain and the murine constant region of an α chain as set forthabove. In this regard, the inventive chimeric TCR can comprise the aminoacid sequence of SEQ ID NO: 17. An α chain of this type can be pairedwith any β chain of a TCR. Preferably, the β chain of the inventivechimeric TCR comprises the human variable region of a β chain and themurine constant region of a β chain as set forth above. In this regard,the inventive chimeric TCR can comprise the amino acid sequence of SEQID NO: 18. The inventive chimeric TCR, therefore, can comprise the aminoacid sequence of SEQ ID NO: 17, SEQ ID NO: 18, or both SEQ ID NOs: 17and 18. Preferably, the inventive chimeric TCR comprises the amino acidsequences of both SEQ ID NOs: 17 and 18.

In an embodiment of the invention, the TCR is a human TCR. The human TCRmay comprise any of the CDR regions as described herein with respect toother aspects of the invention. In this regard, an embodiment of theinvention provides an isolated or purified TCR having antigenicspecificity for HPV 16 E6 and comprising the amino acid sequences of SEQID NOs: 3-8. In another embodiment of the invention, the human TCR maycomprise any of the variable regions described herein with respect toother aspects of the invention.

In an embodiment of the invention, the inventive human TCRs furthercomprise a human constant region. In this regard, the human TCR cancomprise the amino acid sequence of SEQ ID NO: 13 (the constant regionof a human α chain), SEQ ID NO: 14 (the constant region of a human βchain), or both SEQ ID NOs: 13 and 14.

In an embodiment of the invention, the inventive human TCR may comprisea combination of a variable region and a constant region. In thisregard, the TCR can comprise an alpha chain comprising the amino acidsequences of both SEQ ID NO: 9 (the variable region of a human α chain)and SEQ ID NO: 13 (the constant region of a human α chain), a beta chaincomprising the amino acid sequences of both SEQ ID NO: 10 (the variableregion of a human β chain) and SEQ ID NO: 14 (the constant region of ahuman β chain), or all of the amino acid sequences of SEQ ID NOs: 9-10and 13-14.

In an embodiment of the invention, the human TCR can comprise an α chainof a TCR and a β chain of a TCR. Each of the α chain and β chain of theinventive human TCR can independently comprise any amino acid sequence.Preferably, the α chain comprises the human variable region of an αchain and the human constant region of an α chain as set forth above. Inthis regard, the inventive human TCR can comprise the amino acidsequence of SEQ ID NO: 11. An α chain of this type can be paired withany β chain of a TCR. Preferably, the β chain of the inventive human TCRcomprises the human variable region of a β chain and the human constantregion of a β chain as set forth above. In this regard, the inventivehuman TCR can comprise the amino acid sequence of SEQ ID NO: 12. Theinventive human TCR, therefore, can comprise the amino acid sequence ofSEQ ID NO: 11, SEQ ID NO: 12, or both SEQ ID NOs: 11 and 12. Preferably,the inventive human TCR comprises the amino acid sequences of both SEQID NOs: 11 and 12.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein. The term “polypeptide” as used herein includes oligopeptides andrefers to a single chain of amino acids connected by one or more peptidebonds.

With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR (orfunctional variant thereof) of which it is a part, provided that thefunctional portion specifically binds to HPV 16 E6. The term “functionalportion” when used in reference to a TCR (or functional variant thereof)refers to any part or fragment of the TCR (or functional variantthereof) of the invention, which part or fragment retains the biologicalactivity of the TCR (or functional variant thereof) of which it is apart (the parent TCR or parent functional variant thereof). Functionalportions encompass, for example, those parts of a TCR (or functionalvariant thereof) that retain the ability to specifically bind to HPV 16E6 (e.g., in an HLA-A2-dependent manner), or detect, treat, or preventcancer, to a similar extent, the same extent, or to a higher extent, asthe parent TCR (or functional variant thereof). In reference to theparent TCR (or functional variant thereof), the functional portion cancomprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, ormore, of the parent TCR (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCRor functional variant thereof. Desirably, the additional amino acids donot interfere with the biological function of the functional portion,e.g., specifically binding to HPV 16 E6; and/or having the ability todetect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent TCR or functional variant thereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and CDR3 of the variable region(s) of the α chain and/or β chainof a TCR or functional variant thereof of the invention. In anembodiment of the invention, the polypeptide can comprise a functionalportion comprising the amino acid sequence of SEQ ID NO: 3 (CDR1 of αchain), 4 (CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of β chain), 7(CDR2 of β chain), 8 (CDR3 of β chain), or a combination thereof.Preferably, the inventive polypeptide comprises a functional portioncomprising the amino acid sequences of SEQ ID NOs: 3-5; 6-8; or all ofSEQ ID NOs: 3-8. More preferably, the polypeptide comprises a functionalportion comprising the amino acid sequences of all of SEQ ID NOs: 3-8.

In an embodiment of the invention, the inventive polypeptide cancomprise, for instance, the variable region of the inventive TCR orfunctional variant thereof comprising a combination of the CDR regionsset forth above. In this regard, the polypeptide can comprise the aminoacid sequence of SEQ ID NO: 9 (the variable region of an α chain), SEQID NO: 10 (the variable region of aβ chain), or both SEQ ID NOs: 9 and10. Preferably, the polypeptide comprises the amino acid sequences ofboth SEQ ID NOs: 9 and 10.

The inventive polypeptide may further comprise a constant region derivedfrom any suitable species such as, e.g., human or mouse. In this regard,the polypeptide can comprise the amino acid sequence of SEQ ID NO: 13(the constant region of a human α chain), SEQ ID NO: 14 (the constantregion of a human β chain), SEQ ID NO: 15 (the constant region of amurine α chain), SEQ ID NO: 16 (the constant region of a murine (3chain), both SEQ ID NOs: 13 and 14, or both SEQ ID NOs: 15 and 16.Preferably, the polypeptide comprises the amino acid sequences of bothSEQ ID NOs: 13 and 14 or both SEQ ID NOs: 15 and 16.

In an embodiment of the invention, the inventive polypeptide maycomprise a combination of a variable region and a constant region. Inthis regard, the polypeptide can comprise the amino acid sequences ofboth SEQ ID NOs: 9 and 15, both SEQ ID NOs: 9 and 13, both SEQ ID NOs:10 and 16, both SEQ ID NOs: 10 and 14, all of SEQ ID NOs: 9-10 and15-16, or all of SEQ ID NOs: 9-10 and 13-14.

In an embodiment of the invention, the inventive polypeptide cancomprise the entire length of an α or β chain of one of the TCRs orfunctional variant thereof described herein. In this regard, theinventive polypeptide can comprise an amino acid sequence of SEQ ID NOs:11, 12, 17, or 18. Alternatively, the polypeptide of the invention cancomprise α and β chains of the TCRs or functional variants thereofdescribed herein. For example, the inventive polypeptide can comprisethe amino acid sequences of both SEQ ID NOs: 11 and 12 or both SEQ IDNOs: 17 and 18, both SEQ ID NOs: 11 and 18, or both SEQ ID NOs: 17 and12. Preferably, the polypeptide comprises the amino acid sequences ofboth SEQ ID NOs: 11 and 12 or both SEQ ID NOs: 17 and 18.

The invention further provides a protein comprising at least one of thepolypeptides described herein. By “protein” is meant a moleculecomprising one or more polypeptide chains.

In an embodiment, the protein of the invention can comprise a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs: 3-5and a second polypeptide chain comprising the amino acid sequence of SEQID NOs: 6-8. Alternatively or additionally, the protein of the inventioncan comprise a first polypeptide chain comprising the amino acidsequence of SEQ ID NO: 9, and a second polypeptide chain comprising theamino acid sequence of SEQ ID NO: 10. The protein of the invention can,for example, comprise a first polypeptide chain comprising the aminoacid sequence of both SEQ ID NO: 9 and 13 or both SEQ ID NOs: 9 and 15,and a second polypeptide chain comprising the amino acid sequence ofboth SEQ ID NOs: 10 and 14 or both SEQ ID NOs: 10 and 16. The protein ofthe invention can, for example, comprise a first polypeptide chaincomprising the amino acid sequence of SEQ ID NO: 11 or 17, and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 12 or18. In this instance, the protein of the invention can be a TCR.Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising the amino acid sequences of SEQ ID NO: 11and 12 or SEQ ID NO: 17 and 18, or if the first and/or secondpolypeptide chain(s) of the protein further comprise(s) other amino acidsequences, e.g., an amino acid sequence encoding an immunoglobulin or aportion thereof, then the inventive protein can be a fusion protein. Inthis regard, the invention also provides a fusion protein comprising atleast one of the inventive polypeptides described herein along with atleast one other polypeptide. The other polypeptide can exist as aseparate polypeptide of the fusion protein, or can exist as apolypeptide, which is expressed in frame (in tandem) with one of theinventive polypeptides described herein. The other polypeptide canencode any peptidic or proteinaceous molecule, or a portion thereof,including, but not limited to an immunoglobulin, CD3, CD4, CD8, an MHCmolecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

In some embodiments of the invention, the TCRs (and functional portionsand functional variants thereof), polypeptides, and proteins of theinvention may be expressed as a single protein comprising a linkerpeptide linking the α chain and the β chain. In this regard, the TCRs(and functional variants and functional portions thereof), polypeptides,and proteins of the invention comprising the amino acid sequences of SEQID NO: 11 and 12 or SEQ ID NO: 17 and 18 may further comprise a linkerpeptide. The linker peptide may advantageously facilitate the expressionof a recombinant TCR (including functional portions and functionalvariants thereof), polypeptide, and/or protein in a host cell. Thelinker peptide may comprise any suitable amino acid sequence. Forexample, the linker peptide may comprise the amino acid sequence of SEQID NO: 37. Upon expression of the construct including the linker peptideby a host cell, the linker peptide may be cleaved, resulting inseparated α and β chains.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof,can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment, including any of theantibodies and antibody fragments described herein.

Included in the scope of the invention are functional variants of theinventive TCRs described herein. The term “functional variant” as usedherein refers to a TCR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent TCR,polypeptide, or protein, which functional variant retains the biologicalactivity of the TCR, polypeptide, or protein of which it is a variant.Functional variants encompass, for example, those variants of the TCR,polypeptide, or protein described herein (the parent TCR, polypeptide,or protein) that retain the ability to specifically bind to HPV 16 E6for which the parent TCR has antigenic specificity or to which theparent polypeptide or protein specifically binds, to a similar extent,the same extent, or to a higher extent, as the parent TCR, polypeptide,or protein. In reference to the parent TCR, polypeptide, or protein, thefunctional variant can, for instance, be at least about 30%, 50%, 75%,80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acidsequence to the parent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

The TCR (or functional variant thereof), polypeptide, or protein canconsist essentially of the specified amino acid sequence or sequencesdescribed herein, such that other components of the TCR (or functionalvariant thereof), polypeptide, or protein, e.g., other amino acids, donot materially change the biological activity of the TCR (or functionalvariant thereof), polypeptide, or protein. In this regard, the inventiveTCR (or functional variant thereof), polypeptide, or protein can, forexample, consist essentially of the amino acid sequence of SEQ ID NO:11, 12, 17, or 18, both SEQ ID NOs: 11 and 12, both SEQ ID NOs: 17 and18, both SEQ ID NOs: 11 and 18, or both SEQ ID NOs: 17 and 12. Also, forinstance, the inventive TCRs (including functional variants thereof),polypeptides, or proteins can consist essentially of the amino acidsequence(s) of SEQ ID NO: 9, 10, or both SEQ ID NOs: 9 and 10.Furthermore, the inventive TCRs (including functional variants thereof),polypeptides, or proteins can consist essentially of the amino acidsequence of SEQ ID NO: 3 (CDR1 of α chain), SEQ ID NO: 4 (CDR2 of αchain), SEQ ID NO: 5 (CDR3 of α chain), SEQ ID NO: 6 (CDR1 of β chain),SEQ ID NO: 7 (CDR2 of β chain), SEQ ID NO: 8 (CDR3 of β chain), or anycombination thereof, e.g., SEQ ID NOs: 3-5; 6-8; or 3-8.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be of any length, i.e., can compriseany number of amino acids, provided that the TCRs, polypeptides, orproteins (or functional variants thereof) retain their biologicalactivity, e.g., the ability to specifically bind to HPV 16 E6; detectcancer, HPV 16 infection, or HPV-positive premalignancy in a mammal; ortreat or prevent cancer, HPV 16 infection, or HPV-positive premalignancyin a mammal, etc. For example, the polypeptide can be in the range offrom about 50 to about 5000 amino acids long, such as 50, 70, 75, 100,125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or moreamino acids in length. In this regard, the polypeptides of the inventionalso include oligopeptides.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) of the invention can comprise syntheticamino acids in place of one or more naturally-occurring amino acids.Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

The TCR, polypeptide, and/or protein of the invention (includingfunctional variants thereof) can be obtained by methods known in theart. Suitable methods of de novo synthesizing polypeptides and proteinsare described in references, such as Chan et al., Fmoc Solid PhasePeptide Synthesis, Oxford University Press, Oxford, United Kingdom,2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford UniversityPress, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also,polypeptides and proteins can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Green and Sambrook, Molecular Cloning: A LaboratoryManual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.2012; and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, NY, 1994. Further, some ofthe TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be isolated and/or purified from asource, such as a plant, a bacterium, an insect, a mammal, e.g., a rat,a human, etc. Methods of isolation and purification are well-known inthe art. Alternatively, the TCRs, polypeptides, and/or proteinsdescribed herein (including functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, Calif.),Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple PeptideSystems (San Diego, Calif.). In this respect, the inventive TCRs(including functional variants thereof), polypeptides, and proteins canbe synthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive TCRs, polypeptides, orproteins (including any of the functional variants thereof), nucleicacids, recombinant expression vectors, host cells, populations of hostcells, or antibodies, or antigen binding portions thereof. Conjugates,as well as methods of synthesizing conjugates in general, are known inthe art (See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223(2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

An embodiment of the invention provides a nucleic acid comprising anucleotide sequence that encodes any of the TCRs, polypeptides, andproteins herein. By “nucleic acid” as used herein includes“polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” andgenerally means a polymer of DNA or RNA, which can be single-stranded ordouble-stranded, synthesized or obtained (e.g., isolated and/orpurified) from natural sources, which can contain natural, non-naturalor altered nucleotides, and which can contain a natural, non-natural oraltered internucleotide linkage, such as a phosphoroamidate linkage or aphosphorothioate linkage, instead of the phosphodiester found betweenthe nucleotides of an unmodified oligonucleotide. In an embodiment, thenucleic acid comprises complementary DNA (cDNA). It is generallypreferred that the nucleic acid does not comprise any insertions,deletions, inversions, and/or substitutions. However, it may be suitablein some instances, as discussed herein, for the nucleic acid to compriseone or more insertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Green and Sambrook et al., supra, and Ausubel et al., supra.For example, a nucleic acid can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that can be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the TCRs, polypeptides, proteins, or functional functional variantsthereof described herein. In an embodiment of the invention, thenucleotide sequence may comprise, consist, or consist essentially of allof SEQ ID NOs: 31-36 (encoding CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, CDR3β,respectively); all of SEQ ID NOs: 31-33; all of SEQ ID NOs: 34-36; bothof SEQ ID NOs: 19-20 (encoding variable regions of α and β chains,respectively); both of SEQ ID NOs: 23-24 (encoding human constant regionof α and β chains, respectively); both of SEQ ID NOs: 25-26 (encodingmurine constant region of α and β chains, respectively), all of SEQ IDNOs: 19-20 and 23-24, all of SEQ ID NOs: 19-20 and 25-26, both of SEQ IDNOs: 21-22 (encoding human α and β chains, respectively), or both of SEQID NOs: 27-28 (encoding chimeric α and β chains, respectively). Inanother embodiment of the invention, the nucleotide sequence maycomprise, consist, or consist essentially of any one of SEQ ID NOs:19-28 and 31-36.

In an embodiment of the invention, the nucleic acid comprises anon-natural nucleotide sequence. A nucleotide sequence may be consideredto be “non-natural” if the nucleotide sequence is not found in nature.In some embodiments, the nucleotide sequence may be codon-optimized.Without being bound to a particular theory or mechanism, it is believedthat codon optimization of the nucleotide sequence increases thetranslation efficiency of the mRNA transcripts. Codon optimization ofthe nucleotide sequence may involve substituting a native codon foranother codon that encodes the same amino acid, but can be translated bytRNA that is more readily available within a cell, thus increasingtranslation efficiency. Optimization of the nucleotide sequence may alsoreduce secondary mRNA structures that would interfere with translation,thus increasing translation efficiency. In an embodiment of theinvention, the codon-optimized nucleotide sequence may comprise,consist, or consist essentially of SEQ ID NO: 38 (variable region of αchain), SEQ ID NO: 39 (variable region of β chain), or SEQ ID NOs: 38and 39.

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs(including functional portions and functional variants thereof). It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. In an embodiment of the invention, the recombinantexpression vector comprises a nucleotide sequence encoding the α chain,the (3 chain, and linker peptide. For example, in an embodiment, therecombinant expression vector comprises a codon-optimized nucleotidesequence comprising SEQ ID NO: 29 (encoding chimeric α and β chains SEQID NOs: 17 and 18 with a linker positioned between them).

For purposes herein, the term “recombinant expression vector” means agenetically-modified oligonucleotide or polynucleotide construct thatpermits the expression of an mRNA, protein, polypeptide, or peptide by ahost cell, when the construct comprises a nucleotide sequence encodingthe mRNA, protein, polypeptide, or peptide, and the vector is contactedwith the cell under conditions sufficient to have the mRNA, protein,polypeptide, or peptide expressed within the cell. The vectors of theinvention are not naturally-occurring as a whole. However, parts of thevectors can be naturally-occurring. The inventive recombinant expressionvectors can comprise any type of nucleotides, including, but not limitedto DNA and RNA, which can be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors can comprise naturally-occurring,non-naturally-occurring internucleotide linkages, or both types oflinkages. Preferably, the non-naturally occurring or altered nucleotidesor internucleotide linkages does not hinder the transcription orreplication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector. In an especially preferred embodiment, the recombinantexpression vector is an MSGV1 vector. In an embodiment, an MSGV1 vectorcomprising a codon-optimized nucleotide sequence encoding a chimeric TCRcomprising SEQ ID NOs: 17 and 18 of the invention comprises thenucleotide sequence of SEQ ID NO: 30.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Green and Sambrook et al., supra, and Ausubel et al., supra. Constructsof expression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEI, 2μplasmid, λ, SV40, bovine papillomavirus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host cell (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostcell to provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the TCR,polypeptide, or protein (including functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the TCR, polypeptide, or protein(including functional variants thereof). The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5a E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5a cell. For purposes of producing arecombinant TCR, polypeptide, or protein, the host cell is preferably amammalian cell. Most preferably, the host cell is a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell preferablyis a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclearcell (PBMC). More preferably, the host cell is a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. Preferably, the T cell is a human T cell. More preferably,the T cell is a T cell isolated from a human. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells,e.g., Th₁ and Th₂ cells, CD4⁺ T cells, CD8⁺ T cells (e.g., cytotoxic Tcells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g.,central memory T cells and effector memory T cells), naïve T cells, andthe like.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

In an embodiment of the invention, the numbers of cells in thepopulation may be rapidly expanded. Expansion of the numbers of T cellscan be accomplished by any of a number of methods as are known in theart as described in, for example, U.S. Pat. Nos. 8,034,334; 8,383,099;U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J.Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods,128:189-201 (1990).

The invention further provides an antibody, or antigen binding portionthereof, which specifically binds to a functional portion of any of theTCRs (or functional variant thereof) described herein. Preferably, thefunctional portion specifically binds to the cancer antigen, e.g., thefunctional portion comprising the amino acid sequence SEQ ID NO: 3 (CDR1of α chain), 4 (CDR2 of α chain), 5 (CDR3 of α chain), 6 (CDR1 of βchain), 7 (CDR2 of β chain), 8 (CDR3 of β chain), SEQ ID NO: 9 (variableregion of α chain), SEQ ID NO: 10 (variable region of β chain), or acombination thereof, e.g., 3-5; 6-8; 3-8; 9; 10; or 9-10. Morepreferably, the functional portion comprises the amino acid sequences ofSEQ ID NOs: 3-8 or SEQ ID NOs: 9 and 10. In a preferred embodiment, theantibody, or antigen binding portion thereof, binds to an epitope whichis formed by all 6 CDRs (CDR1-3 of the alpha chain and CDR1-3 of thebeta chain). The antibody can be any type of immunoglobulin that isknown in the art. For instance, the antibody can be of any isotype,e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal orpolyclonal. The antibody can be a naturally-occurring antibody, e.g., anantibody isolated and/or purified from a mammal, e.g., mouse, rabbit,goat, horse, chicken, hamster, human, etc. Alternatively, the antibodycan be a genetically-engineered antibody, e.g., a humanized antibody ora chimeric antibody. The antibody can be in monomeric or polymeric form.Also, the antibody can have any level of affinity or avidity for thefunctional portion of the inventive TCR (or functional variant thereof).Desirably, the antibody is specific for the functional portion of theinventive TCR (or functional variants thereof), such that there isminimal cross-reaction with other peptides or proteins.

Methods of testing antibodies for the ability to bind to any functionalportion or functional variant of the inventive TCR are known in the artand include any antibody-antigen binding assay, such as, for example,radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., infra, andU.S. Patent Application Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8^(th) Ed., Garland Publishing, New York,N.Y. (2011)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1.

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Green andSambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^(th)Edition, Cold Spring Harbor Laboratory Press, New York (2012)). Phageencoding a variable region with the desired specificity are selected forspecific binding to the desired antigen, and a complete or partialantibody is reconstituted comprising the selected variable domain.Nucleic acid sequences encoding the reconstituted antibody areintroduced into a suitable cell line, such as a myeloma cell used forhybridoma production, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in, for example, U.S. Pat. No. 5,639,641 and Pedersen et al.,J. Mol. Biol., 235, 959-973 (1994).

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)2, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

The inventive TCRs, polypeptides, proteins, (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), can be isolated and/or purified. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. For example, the purity can beat least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or canbe 100%.

The inventive TCRs, polypeptides, proteins (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), all of which are collectively referred to as“inventive TCR materials” hereinafter, can be formulated into acomposition, such as a pharmaceutical composition. In this regard, theinvention provides a pharmaceutical composition comprising any of theTCRs, polypeptides, proteins, functional portions, functional variants,nucleic acids, expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof)described herein, and a pharmaceutically acceptable carrier. Theinventive pharmaceutical compositions containing any of the inventiveTCR materials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as a chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the particular inventive TCR material underconsideration. Such pharmaceutically acceptable carriers are well-knownto those skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Suitable formulations may include any of those for oral,parenteral, subcutaneous, intravenous, intramuscular, intraarterial,intrathecal, or interperitoneal administration. More than one route canbe used to administer the inventive TCR materials, and in certaininstances, a particular route can provide a more immediate and moreeffective response than another route.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered should be sufficient to effect,e.g., a therapeutic or prophylactic response, in the subject or animalover a reasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR (or functional variant or functionalportion thereof), polypeptide, or protein upon administration of a givendose of such T cells to a mammal among a set of mammals of which is eachgiven a different dose of the T cells, could be used to determine astarting dose to be administered to a mammal. The extent to which targetcells are lysed or IFN-γ is secreted upon administration of a certaindose can be assayed by methods known in the art.

The dose of the inventive TCR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive TCR material.Typically, the attending physician will decide the dosage of theinventive TCR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive TCR material to beadministered, route of administration, and the severity of the conditionbeing treated. In an embodiment in which the inventive TCR material is apopulation of cells, the number of cells administered per infusion mayvary, e.g., from about 1×10⁶ to about 1×10¹² cells or more.

One of ordinary skill in the art will readily appreciate that theinventive TCR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive TCR materials is increased through the modification. Forinstance, the inventive TCR materials can be conjugated either directlyor indirectly through a bridge to a targeting moiety. The practice ofconjugating compounds, e.g., inventive TCR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the inventive TCR materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “bridge” as used herein, refers to any agent or molecule thatlinks the inventive TCR materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive TCRmaterials, which are not necessary for the function of the inventive TCRmaterials, are ideal sites for attaching a bridge and/or a targetingmoiety, provided that the bridge and/or targeting moiety, once attachedto the inventive TCR materials, do(es) not interfere with the functionof the inventive TCR materials, i.e., the ability to bind to HPV 16 E6;or to detect, treat, or prevent cancer, HPV 16 infection, orHPV-positive premalignancy.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional variants thereof), polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, or populations ofcells can be used in methods of treating or preventing cancer, HPV 16infection, or HPV-positive premalignancy. Without being bound to aparticular theory, the inventive TCRs (and functional variants thereof)are believed to bind specifically to HPV 16 E6, such that the TCR (orrelated inventive polypeptide or protein and functional variantsthereof), when expressed by a cell, is able to mediate an immuneresponse against a target cell expressing HPV 16 E6. In this regard, theinvention provides a method of treating or preventing a condition in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, TCRs (and functional variants thereof), polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs (andfunctional variants thereof), polypeptides, proteins described herein,or any host cell or population of cells comprising a recombinant vectorwhich encodes any of the TCRs (and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and detectingthe complex, wherein detection of the complex is indicative of thepresence of the condition in the mammal, wherein the condition iscancer, HPV 16 infection, or HPV-positive premalignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs (and functionalvariants thereof), polypeptides, proteins, nucleic acids, recombinantexpression vectors, host cells, populations of cells, or antibodies, orantigen binding portions thereof, described herein, can be labeled witha detectable label such as, for instance, a radioisotope, a fluorophore(e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme(e.g., alkaline phosphatase, horseradish peroxidase), and elementparticles (e.g., gold particles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer,cancer of the anus, anal canal, or anorectum, cancer of the eye, cancerof the intrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vagina, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynxcancer, liver cancer, lung cancer, malignant mesothelioma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer ofthe oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer, skin cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is HPV 16-positive cancer. While thecancers most commonly associated with HPV 16 infection include cancer ofthe uterine cervix, oropharynx, anus, anal canal, anorectum, vagina,vulva, and penis, the inventive methods may be used to treat any HPV16-positive cancer, including those that occur at other anatomicalareas.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the isolation of human anti-HPV 16 TCRs fromtumor.

A sample of a metastatic HPV 16 E6-positive anal cancer tumor (tumor3809) was obtained from a patient. The tumor sample was analyzed forexpression of HPV 16 E6, HPV 16 E7, HPV 18 E6, and HPV 18 E7 relative toglyceraldehyde 3-phosphate dehydrogenase (GAPDH) by reversetranscriptase (RT) polymerase chain reaction (PCR). Relative expressionof HPV 16 E6, HPV 16 E7, HPV 18 E6, and HPV 18 E7 was compared to thatof CaSki cells, HeLa cells, and 624 cells (melanoma cell line). Theresults are shown in FIG. 1. As shown in FIG. 1, the tumor 3809 samplewas positive for HPV 16 E6 expression.

The tumor 3809 sample was divided into 24 fragments and tumorinfiltrating lymphocytes (TIL) were obtained from the various fragments.The TIL were co-cultured in a 96-well plate with autologous immaturedendritic cells (DCs) which had been pulsed with HPV 16 E6 alone, HPV 16E6 in combination with anti-class I antibody, HPV 16 E7 alone, HPV 16 E7in combination with anti-class I antibody, gp100, or OKT3.Interferon-gamma (IFN-γ) was measured. The results are shown in FIGS. 2Aand 2B. As shown in FIGS. 2A and 2B, TIL were reactive against HPV 16 E6but not gp100 or E7. The anti-HPV 16 E6 reactivity of the TIL wasblocked by anti-class I antibody.

Cells from the reactive co-culture wells were selected using anti-4-1BBmagnetic beads. Rapid expansion of the numbers of selected cells wasperformed using the Rapid Expansion Protocol (REP) as previouslydescribed (Dudley et al. J. Immunother. 26:332-42 (2003) and Riddell etal. J. Immunol. Methods 128:189-201 (1990)). Briefly, TIL were culturedwith irradiated (40 Gy) allogeneic peripheral blood mononuclear “feeder”cells in complete medium (CM) with 30 ng/mL anti-CD3 antibody and 6000IU/mL IL-2.

The expanded numbers of 3809 4-1BB-selected cells were co-cultured with293-A2 cells (HEK-293 cells with stable expression of HLA-A2)transfected with green fluorescent protein (GFP), 293-A2 cellstransfected with E6, 3809 lymphoblastoid cell line (LCL) (B cells thathave been transformed using Epstein-Barr virus) cultured withoutpeptide, 3809 LCL co-cultured with an HPV 16 E6 peptide pool, or OKT3.The peptide pool included 15-mer peptides with 11-amino-acid overlapsthat covered the complete sequence of HPV 16 E6. IFN-γ was measured. Theresults are shown in FIG. 3. As shown in FIG. 3, the expanded numbers ofTIL were reactive against 293-A2 cells transfected with E6 but not293-A2 cells transfected with GFP. The 3809 LCL cells co-cultured withthe E6 peptide pool demonstrated reactivity while the 3809 LCL cellsco-cultured with no peptides did not. Flow cytometry studies showed thatthe expanded numbers of cells bound to HLA-A2/E6₂₉₋₃₈ tetramer.

Cells were further selected by sorting using anti-4-1BB magnetic beadswithout further cycles of REP or cloning followed by 5′ RapidAmplification of cDNA Ends (RACE). A genotype analysis of the 5′ RACEproducts from the magnetic bead isolation is shown in Table A. As shownin Table A, a nearly clonal population of cells was obtained.

TABLE A TRAV TRAJ Colonies TRBV TRBJ TRBD Colonies TRAV35*02 TRAJ41*01 4TRBV7- TRBV2- TRBD1*01 8 6*01 3*01 TRAV10*01 TRAJ44*01 3 TRBV14*01TRBJ1- TRBD1*01 1 6*01 TRAV5*01 TRAJ34*01 1

A nucleotide sequence comprising cDNA (SEQ ID NO: 21) encoding an alphachain comprising the amino acid sequence of SEQ ID NO: 11 was obtainedfrom TRAV35*02/TRAJ41*01. A nucleotide sequence comprising cDNA (SEQ IDNO: 22) encoding a beta chain comprising the amino acid sequence of SEQID NO: 12 was obtained from TRBV7-6*01/TRBV2-3*01/TRBD1*01.

Example 2

This example demonstrates a method of making a chimeric anti-HPV 16 TCRcomprising a human variable region and a mouse constant region.

A nucleotide sequence encoding a chimeric TCR including a mouse constantregion and a human variable region was prepared as follows. Thenucleotide sequences encoding the original (human) constant regions ofthe alpha and beta chains of the TCR obtained in Example 1 (constantregion amino acid sequences of SEQ ID NOs: 23 and 24, respectively) wereexcised and replaced with nucleotide sequences encoding a murineconstant region of the alpha and beta chains, respectively. Theresulting nucleotide sequences encoding the chimeric alpha and betachains were cloned into a single nucleotide sequence with a nucleotidesequence encoding a picornavirus 2A peptide positioned between the alphaand beta chains. The combined nucleotide sequence was codon-optimized(opt) for expression in human tissues to provide a vector insert (SEQ IDNO: 29). The vector insert was cloned into an MSGV1 expression vectorresulting in the nucleotide sequence of SEQ ID NO: 30 (E6 TCR). The TCRencoded by the vector comprised an alpha chain comprising an amino acidsequence comprising SEQ ID NO: 17 and a beta chain comprising an aminoacid sequence comprising SEQ ID NO: 18.

Example 3

This example demonstrates that peripheral blood lymphocytes (PBL)transduced with a recombinant expression vector encoding the amino acidsequences of SEQ ID NOs: 17 and 18 specifically recognize HPV16-positive tumor cell lines in an HLA-A2-restricted manner.

Peripheral blood lymphocytes (PBL) were transduced with the expressionvector of Example 2 and were co-cultured with target 293-A2 cells pulsedwith HPV 16 E6₂₉₋₃₈ peptide, 293-A2 cells pulsed with HPV 16 E7₁₁₋₁₉peptide, 293-A2 cells transduced with a plasmid encoding HPV 16 E6,293-A2 cells transduced with a plasmid encoding GFP, 293 cellstransduced with a plasmid encoding HPV 16 E6, 624 cells transduced witha plasmid encoding HPV 16 E6, 624 cells transduced with a plasmidencoding HPV 16 E7, SCC152 cells, SCC90 cells, CaSki cells, HPV-18cervical cancer cells, melanoma control cells, cholangio control cells,624 cells, or SiHa cells. IFN-γ was measured. The results are shown inFIG. 4A. As shown in FIG. 4A, PBMC transduced with a recombinantexpression vector encoding the amino acid sequences of SEQ ID NOs: 17and 18 specifically recognizes HPV 16-positive tumor cell lines andother HLA-A2⁺ HPV16⁺ targets in an HLA-A2-restricted manner.

PBL transduced with the expression vector of Example 2 were co-culturedwith target 293-A2 cells transduced with a plasmid encoding E6, 293cells transduced with a plasmid encoding E6, SCC90 cells, CaSki cells,or 624 cells (melanoma cell line) with no antibody, anti-MHC Class Iantibody, or anti-MHC Class II antibody. DMF5 (T cells transduced toexpress a MHC class I-restricted TCR against MART-1) were co-culturedwith a melanoma cell line (624) that is recognized by DMF5 with noantibody, anti-MHC Class I antibody, or anti-MHC Class II antibody.4.7.20 (T cells transduced to express a MHC class II-restricted TCRagainst HPV 16 E7) were cultured with PBMC pulsed with the E7 peptidepool “E7 peptides” with no antibody, anti-MHC Class I antibody, oranti-MHC Class II antibody. IFN-γ was measured. The results are shown inFIG. 4B. As shown in FIG. 4B, anti-MHC Class I antibody blocked thereactivity of the transduced cells against HLA-A2⁺ HPV16⁺ targets, whileanti-Class II antibody did not block reactivity.

Example 4

This example demonstrates that cells transduced with a recombinantexpression vector encoding the amino acid sequences of SEQ ID NOs: 17and 18 bind to HLA-A2-E6₂₉₋₃₈ tetramer in a CD8-independent manner.

PBL transduced with the recombinant expression vector of Example 2 wassorted into CD8-positive cells and CD8-negative cells by FACS. Bindingto HLA-A2-E6₂₉₋₃₈ tetramer was measured by flow cytometry. CD8-positiveand CD8-negative cells both bound to HLA-A2-E6₂₉₋₃₈ tetramer.

Example 5

This example demonstrates that CD4 and CD8-positive cells transducedwith a recombinant expression vector encoding the amino acid sequencesof SEQ ID NOs: 17 and 18 specifically recognize HPV-16 positive tumorcell lines.

CD8-positive or CD4-positive PBL were not transduced (untransduced) ortransduced with the expression vector of Example 2 and were co-culturedwith target 293-A2 cells pulsed with HPV 16 E6₂₉₋₃₈ peptide, 293-A2cells pulsed with HPV 16 E7₁₁₋₁₉ peptide, 293-A2 cells transduced with aplasmid encoding HPV 16 E6, 293-A2 cells transduced with a plasmidencoding GFP, 293-A2 cells, retrovirus-transduced 624 cells stablyexpressing HPV 16 E7 (624-E7), retrovirus-transduced 624 cells stablyexpressing HPV 16 E6 (624-E6), SCC152 cells, SCC90 cells, CaSki cells,HPV-18 cervical cancer cells, melanoma control cells, 624 cells, or SiHacells. IFN-γ was measured. The results are shown in FIG. 5. As shown inFIG. 5, CD8 positive and CD4 positive PBMC transduced with a recombinantexpression vector encoding the amino acid sequences of SEQ ID NOs: 17and 18 both specifically recognize HPV 16-positive tumor cell lines andother HLA-A2⁺ HPV16⁺ targets in an HLA-A2-restricted manner.

Example 6

This example demonstrates that cells transduced with a recombinantexpression vector encoding the amino acid sequences of SEQ ID NOs: 17and 18 demonstrate avid recognition of HPV 16 E6₂₉₋₃₈-pulsed T2 cells.

CD8-positive or CD4-positive PBL were not transduced (untransduced) ortransduced with the expression vector of Example 2 and were co-culturedwith target T2 cells pulsed with varying concentrations of HPV 16E6₂₉₋₃₈ peptide. IFN-γ was measured. The results are shown in FIGS. 6Aand 6B. As shown in FIGS. 6A and 6B, CD4 positive and CD8 positive cellstransduced with a recombinant expression vector encoding the amino acidsequences of SEQ ID NOs: 17 and 18 demonstrated avid recognition of HPV16 E6₂₉₋₃₈-pulsed T2 cells.

Example 7

This example demonstrates a method of treating HPV 16⁺ cancer in a humanpatient, comprising administering to the patient autologous T cellstransduced to express an anti-HPV 16 E6₂₉₋₃₈ TCR comprising the aminoacid sequences of SEQ ID NOs: 17 and 18.

Patients will have recurrent/refractory or metastatic HPV-16⁺ cancer. Asample of cancerous tissue will be tested for HPV 16 genotype by in situhybridization (ISH) or PCR. Patients will also be tested for HLA-A2expression. The patients will have had a prior first line treatment forrecurrent/refractory or metastatic disease, or the patient will havedeclined standard therapy.

Patients will be treated with cyclophosphamide (60 mg/kg/dayintravenously (IV)) on days −7 and −6 and fludarabine (25 mg/m2/day IV)on days −5 through −1. Autologous PBMC will be transduced with the MSGV1expression vector of Example 2. The numbers of transduced cells will berapidly expanded as previously described (Dudley et al. J. Immunother.26:332-42 (2003) and Riddell et al. J. Immunol. Methods 128:189-201(1990)). Briefly, cells will be cultured with irradiated (40 Gy)allogeneic peripheral blood mononuclear “feeder” cells in completemedium (CM) with 30 ng/mL anti-CD3 antibody and 6000 IU/mL IL-2.Expanded numbers of transduced cells will be administered to thepatients along with a high dose of interleukin (IL)-2 on day 0.

Objective tumor responses will be evaluated according to RECIST(Response Evaluation Criteria In Solid Tumors) 1.0. If at least threeout of 18 patients respond to treatment at four months or more aftertreatment, the cohort will be expanded to 35 patients. Toxicity willalso be evaluated. Immunological studies (including, for example,expansion, persistence, phenotype, and function of the infused cells)will also be studied.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-41. (canceled)
 42. An isolated or purified nucleic acid comprising anucleotide sequence encoding a T cell receptor (TCR) having antigenicspecificity for human papillomavirus (HPV) 16 E6 and comprising: (a) ahuman variable region and a murine constant region; and/or (b) the aminoacid sequences of SEQ ID NOs: 3-8.
 43. The isolated or purified nucleicacid of claim 42, wherein the TCR has antigenic specificity for HPV 16E6₂₉₋₃₈ comprising the amino acid sequence of SEQ ID NO:
 2. 44. Theisolated or purified nucleic acid of claim 42, wherein the TCR comprisesthe amino acid sequences of SEQ ID NOs: 15 and
 16. 45. The isolated orpurified nucleic acid of claim 42, wherein the TCR comprises the aminoacid sequences of SEQ ID NOs: 17 and
 18. 46. The isolated or purifiednucleic acid of claim 42, wherein the TCR comprises the amino acidsequences of SEQ ID NOs: 9 and
 10. 47. The isolated or purified nucleicacid of claim 42, wherein the TCR comprises the amino acid sequences ofSEQ ID NOs: 13 and
 14. 48. The isolated or purified nucleic acid ofclaim 42, wherein the TCR comprises the amino acid sequences of SEQ IDNOs: 11 and
 12. 49. An isolated or purified nucleic acid comprising anucleotide sequence encoding a protein comprising polypeptide(s),wherein the polypeptide(s) comprise(s) a functional portion of the TCRof claim 42, wherein the functional portion comprises the amino acidsequences of (a) SEQ ID NOs: 3-5, (b) SEQ ID NOs: 6-8, or (c) SEQ IDNOs: 3-8.
 50. The isolated or purified nucleic acid according to claim49, wherein the protein comprises a first polypeptide chain comprisingthe amino acid sequences of SEQ ID NOs: 3-5 and a second polypeptidechain comprising the amino acid sequences of SEQ ID NOs: 6-8.
 51. Theisolated or purified nucleic acid according to claim 49, wherein theprotein comprises a first polypeptide chain comprising the amino acidsequence of SEQ ID NO: 9 and a second polypeptide chain comprising theamino acid sequence of SEQ ID NO:
 10. 52. The isolated or purifiednucleic acid according to claim 49, wherein the protein comprises afirst polypeptide chain comprising the amino acid sequence of SEQ ID NO:11 and a second polypeptide chain comprising the amino acid sequence ofSEQ ID NO:
 12. 53. The isolated or purified nucleic acid of claim 49,wherein the protein comprises a first polypeptide chain comprising theamino acid sequence of SEQ ID NO: 17 and a second polypeptide chaincomprising the amino acid sequence of SEQ ID NO:
 18. 54. The isolated orpurified nucleic acid of claim 49, wherein the protein is a fusionprotein.
 55. The isolated or purified nucleic acid of claim 49, whereinthe protein is a recombinant antibody.
 56. The nucleic acid according toclaim 42, wherein the nucleotide sequence is codon-optimized.
 57. Arecombinant expression vector comprising the nucleic acid according toclaim
 42. 58. An isolated host cell comprising the recombinantexpression vector of claim
 57. 59. The host cell according to claim 58,wherein the cell is human.
 60. A population of cells comprising at leastone host cell of claim
 58. 61. A pharmaceutical composition comprisingthe population of cells of claim 60 and a pharmaceutically acceptablecarrier.